The physiological role of the mitochondrial folate enzyme ALDH1L2, discovered by the PI's lab in 2010, is not well understood. This enzyme catalyzes the reaction: NADP+ + 10-formyltetrahydrofolate ? NADPH + CO2 + tetrahydrofolate, which can be important for the mitochondrial NADPH production from the oxidation of folate- bound one-carbon groups. In support of this function, our recent gene knockout experiments show that deletion of Aldh1l2 in mice causes increased oxidative stress. Furthermore, Aldh1l2-/- mice develop ulcerative dermatitis, and have highly enlarged (4-fold) spleen and extensively altered metabotype. The mechanistic basis underlying these phenotypes is currently unknown but has clinical relevance given the recent identification of patients with ALDH1L2 mutations who were diagnosed with a rare neurocutaneous disease or autistic spectrum disorder. Our metabolomics analysis of patient's fibroblasts identified dramatic changes in the cellular lipid repertoire, the outcome most likely caused by the impairment of mitochondrial CoA-dependent fatty acid metabolism. This leads to mitochondrial dysfunction, which is manifested as insufficient energy production and oxidative stress. In further support of this mechanism, fibroblasts from ALDH1L2-deficient patients have fragmented mitochondria and show the accumulation of lipid droplets. Importantly, the restoration of the ALDH1L2 enzyme in these fibroblasts rescues the phenotype and metabotype, making these cells similar to fibroblasts from healthy individuals. Based on these findings, we hypothesize that ALDH1L2 serves distinct metabolic function in the cell, the maintenance of redox state through the mitochondrial NADPH generation linked to the folate cycle. The loss of ALDH1L2 leads to mitochondrial dysfunction, energy disbalance and oxidative stress. Thus, deleterious mutations in the ALDH1L2 gene are underlying cause of mitochondria-related human disorders/diseases. This proposal will determine the role of ALDH1L2 in cellular metabolism and will link its mutations as causative factor of human diseases through the following aims: (1) Test the hypothesis that ALDH1L2 maintains mitochondrial redox balance and controls coenzyme A biosynthesis and fatty acid oxidation. (2) Define the role of ALDH1L2 in partitioning of one-carbon groups between energy production or mitochondria to cytoplasm shuttling. (3) Establish ALDH1L2 deficiency as the cause of rare genetic disorders in humans and explore underlying mechanisms. It is now clear that mitochondrial dysfunction is an important component in the pathophysiology of numerous diseases that had not been previously identified. The role of ALDH1L2 in maintenance of mitochondrial function, as well as in mitochondria-related diseases, is largely unexplored. This proposal will fill this knowledge gap and will provide mechanistic insight into the role of ALDH1L2 in human diseases.